膜におけるビタミンEの抗酸化ダイナミクス

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Dynamics of the initiation of lipid peroxidation in membranes and the inhibition of it by α-tocopherol (α-Toc) were investigated under the biological condition. (1) First, we studied the initiation of lipid peroxidation in phosphatidylcholine (PC) liposomes. Membrane lipid peroxidation was not directly induced by superoxide (O_2^-), H_2O_2, and OH-radical (・OH) itself. The initiation of O_2^--dependent peroxidation required the presence of preformed PC-hydroperoxides (PC-OOH) and chelated metal catalyst such as Fe^<3+>-NTA (nitrilotriacetate). We proposed the mechanism that a radical group of PC-alkoxy radical (PC-O・), which was derived from degraded PC-OOH by the reaction with reduced Fe^<3+>-NTA by O_2^-, penetrates from membrane surface into the inner hydrophobic region and transfers "the initiation message of lipid peroxidation". (2) Second, we studied the location of α-Toc in liposome membranes, and the dynamics of its radical trapping and recycling by ascorbic acid (AsA). Little interaction of α-Toc with acrylamide, a water-soluble fluorescence quencher with a very low capacity to penetrate into the phospholipid membranes, suggested little exposure of the OH-group of α-Toc at the membrane surface. The oxidation rate of α-Toc by positively charged Fe^<3+> was 150 times slower in negatively charged liposomes than in negatively charged SDS micelles, indicating that less than 0.65 mol% of the OH-groups of α-Toc was probably exposed at the membrane surface. The dynamic quenching abilities of n-(N-oxy-4,4'-dimethyloxazolidine-2-yl)stearic acids (n-NS) labeled at different depths of membranes on the intrinsic fluorescence of α-Toc were in the order 5-NS>7-NS>12-NS>16-NS, indicating that the OH-group of α-Toc is located most at a position corresponding to 5-methylene carbon-inner from the surface and a little in hydrophobic region. AsA in bulk water phase completely suppressed the consumption of α-Toc during lipid peroxidation induced by 2,2'-azobis-(dimethylvaleronitrite) (AMVN), a hydrophobic radical precursor, in negatively charged liposomes, though negatively charged AsA could not penetrate into negatively and neutrally charged membranes (the ESR spectra of 5-NS and 16-NS labeled in these liposomes were no changed by the addition of AsA). These findings indicate that α-Toc is oxidized at inner region of the membranes by lipid radicals, and then floats up to the surface, where it is regenerated to α-Toc by AsA. (3) Finally, we measured the rate constant (k_s) of α-Toc for deactivating singlet oxygen (^10_2), which was generated by photoirradiation at the membrane surface, hydrophobic inner region and in bulk water. The following three factors ((1) the concentration and (2) mobility of α-Toc in membranes and of its active moiety at the membrane domains, and (3) the dielectric constant at membrane domains) are experimentally confirmed to be important for the consideration of k_s value of α-Toc in membranes: (1) the concentration of α-Toc, which was higher in membrane than in EtOH solution; the local concentration of the active moiety (OH-group) in membranes, which was 0%, 50-60%, and 40-50% at membrane surface polar zone (PZ), inner hydrogen belt (HB) and hydrophobic core (HC), (2) the mobility of α-Toc, which was higher in EtOH solution than in membranes, and higher at liquid crystalline state than at gel state of membrane phospholipids; the mobility of α-Toc, which was higher than that of β-carotene, because α-Toc locates at one half of the bilayer membrane but β-carotene locates across the bilayer; the local mobility of the OH-group of α-Toc, which was higher at HC region than at HB region, (3) the dielectric constant (micropolarity:ε), which reflects the reactivity of OH-group of α-Toc and ^10_2, at the membrane domains (εwas higher at HB region than at HC regions).
2007-10-25

膜におけるビタミンEの抗酸化ダイナミクス

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